The majority of antiviral restriction factors are interferon (IFN) inducible, and are thus collectively referred to as IFN-stimulated genes (ISGs); many, however, are NOT directly regulated by IFN and remain poorly characterized. Notably, some of these cellular factors are known to modulate lipids and/or membrane properties, thereby disrupting the replication of HIV and other viruses. Two recent examples from this category are TIM (T- cell immunoglobulin and mucin domain) and SERINC (serine incorporator) family proteins, which directly interact with or possibly regulate the synthesis of phosphatidylserine (PS), thus inhibiting HIV release or infectivity. Interestingly, our preliminary data and two recent reports published in Nature showed that the lentiviral Nef proteins effectively antagonize the restriction by TIMs and SERINCs. Moreover, we have recently observed that SERINC proteins potentiate the ability of TIM-1 to block HIV-1 release and that SERINCs do this by stabilizing the TIM expression in the viral producer cells. In this application, we propose to test several novel hypotheses that address the possible link between TIM, SERINC, PS and Nef. Aim 1 will determine how HIV-1 Nef antagonizes TIM-mediated inhibition of viral release through modulating the synthesis and trafficking of TIM-1 and PS. Aim 2 will focus on understanding of the role of endogenous SERINC proteins in CD4+ T cells that regulates the TIM expression and stability, as well as in modulating lipids in the viral producer cell and viral particles, collectively contributing to the inhibition of HIV-1 release and replication. Aim 3 will define the molecular interplay between SERINC and TIM proteins in viral producer cells, and dissect how HIV-1 Nef protein down- modulates this process to promote HIV-1 production and infection. Results from the proposed experiments will provide novel and unified mechanistic insights into the interplay between TIM, SERINC and HIV Nef, and will enhance our understanding of virus-host interaction and AIDS pathogenesis.